Effects of single synonymous substitutions on folding efficiency demonstrate the influence of rare codons and protein structure

Proteins can misfold during cotranslational folding, but how codon sequences influence this process is not well understood. Here, we develop an in vivo assay to comprehensively study the impact of single synonymous substitutions on protein folding efficiency and apply it to the N-terminal domain of E. Coli protein ddlA. We map the influence of codons along the sequence and demonstrate that codons can substantially influence the folding efficiency and that the impact depends on the structure and topology of the protein. A cluster of codons associated with residues in the center of the domain fold strongly influences the folding efficiency. Moreover, substitutions to rarer codons generally lead to increased folding efficiency. A mRNA sequence exclusively made up of rare codons results in higher expression than one with only common codons. Our results highlight the importance of rare codons in cotranslational folding and the relationship between codon sequence and protein structure.